Recombinant expression of
The present invention relates to methods for the recombinant expression of Chlamydia major outer membrane protein (MOMP) comprising transforming a population of E. coli host cells with an expression vector comprising a nucleic acid molecule that encodes Chlamydia MOMP and encodes a leader sequence for targeting the MOMP to the outer membrane of the cell, wherein the nucleic acid molecule is operatively linked to a promoter. The method of the invention allows expression of MOMP in the outer membrane of the cell, which leads to protein folding that is more like native MOMP relative to a MOMP protein that is expressed intracellularly. Also provided by the invention are uses of the recombinant MOMP in pharmaceutical compositions and methods for the treatment and/or prophylaxis of Chlamydia infection and/or the effects thereof.
1. A method for the recombinant expression of Chlamydia major outer membrane protein (MOMP) comprising: (a) transforming a population of Escherichia coli ( E. coli ) host cells with an expression vector comprising a nucleic acid molecule comprising a sequence of nucleotides that encode a leader sequence for targeting the MOMP to the outer membrane of the cell and a sequence of nucleotides that encode chlamydia MOMP, wherein the nucleic acid molecule is operatively linked to a promoter; (b) culturing the transformed cells under conditions that permit expression of the nucleic acid molecule and translocation to the outer membrane of the cells to produce a recombinant Chlamydia MOMP; and (c) optionally purifying the MOMP; wherein the sequence of nucleotides that encode the MOMP is codon harmonized or codon optimized for optimal expression in an E. coli host cell and the expression vector is a vector that is associated with a low to moderate transcription or translation rate.
2. The method of claim 1 , wherein the leader sequence is operatively linked to the N-terminus of the MOMP.
3. The method of claim 1 , wherein the leader sequence is directly adjacent to the MOMP sequence.
4. The method of claim 1 , wherein the nucleic acid molecule further comprises a sequence of nucleotides that encodes a polypeptide attached to the MOMP, wherein the polypeptide is selected from the group consisting of: a linker, an additional antigen, a polypeptide having adjuvant properties, a polypeptide for facilitating purification, a polypeptide for enhancing stability of the MOMP, a carrier protein, and a marker protein.
5. The method of claim 1 , wherein the MOMP comprises a sequence of amino acids as set forth in any of SEQ ID NO: 23 and 25-31.
6. The method of claim 1 , wherein the leader sequence comprises the amino acid sequence of pectate lysase B (PelB) sequence set forth in SEQ ID NO:13.
7. The method of claim 6 , wherein the expression vector comprises a low or moderate strength promoter.
8. The method of claim 7 , wherein the method further comprises a step of inducing the transformed host cell with isopropyl β-D-1-thiogalactopyranoside (IPTG) for from about 4 hours to about 6 hours.
9. The method of claim 8 , wherein the induction step is carried out at about 30° C.
10. The method of claim 8 , wherein the cell density (OD590) is allowed to reach about 0.4 to about 0.8 before the induction step is carried out.
11. A method for the recombinant expression of Chlamydia major outer membrane protein (MOMP) comprising:
(a) transforming a population of Escherichia coli ( E. coli ) host cells with an expression vector comprising a nucleic acid molecule comprising a sequence of nucleotides that encode a leader sequence for targeting the MOMP to the outer membrane of the cell and a sequence of nucleotides that encode chlamydia MOMP, wherein the nucleic acid molecule is operatively linked to a promoter;
(b) culturing the transformed cells under conditions that permit expression of the nucleic acid molecule and translocation to the outer membrane of the cells to produce a recombinant Chlamydia MOMP; and
(c) optionally purifying the MOMP;
wherein the leader sequence comprises the amino acid sequence of the pectate lysase B (PelB) sequence set forth in SEQ ID NO: 13.
12. The method of claim 11 , wherein the sequence of nucleotides that encodes the MOMP is codon harmonized or codon optimized for optimal expression in an E. coli host cell.
13. The method of claim 11 , wherein the leader sequence is operatively linked to the N-terminus of the MOMP.
14. The method of claim 11 , wherein the leader sequence is directly adjacent to the MOMP sequence.
15. The method of claim 11 , wherein the nucleic acid molecule further comprises a sequence of nucleotides that encodes a polypeptide attached to the MOMP, wherein the polypeptide is selected from the group consisting of: a linker, an additional antigen, a polypeptide having adjuvant properties, a polypeptide for facilitating purification, a polypeptide for enhancing stability of the MOMP, a carrier protein, and a marker protein.
16. The method of claim 11 , wherein the expression vector comprises a low or moderate strength promoter.
17. The method of claim 11 , wherein the expression vector is a vector that is associated with a low to moderate transcription or translation rate.
18. The method of claim 11 , wherein the method further comprises a step of inducing the transformed host cell with isopropyl β-D-1-thiogalactopyranoside (IPTG) for from about 4 hours to about 6 hours.
19. The method of claim 18 , wherein the induction step is carried out at about 30° C.
20. The method of claim 18 , wherein the cell density (OD590) is allowed to reach about 0.4 to about 0.8 before the induction step is carried out.